Cell proliferation and programmed cell death play important roles in the growth and development of an organism. In proliferative diseases such as cancer, the processes of cell proliferation and/or programmed cell death are often perturbed. For example, a cancer cell may have unregulated cell division through either the overexpression of a positive regulator of the cell cycle or the loss of a negative regulator of the cell cycle, perhaps by mutation. Alternatively, a cancer cell may have lost the ability to undergo programmed cell death through the overexpression of a negative regulator of apoptosis. Therefore, there is a need to develop new therapeutic agents that will restore the processes of checkpoint control and programmed cell death to cancerous cells.
RNA interference (RNAi) is an evolutionarily conserved process in which recognition of double-stranded RNA (dsRNA) ultimately leads to posttranscriptional suppression of gene expression. This suppression is mediated by short double stranded RNA (dsRNA), also called small interfering RNA (siRNA), which induces specific degradation of mRNA through complementary base pairing. In several model systems, i.e., mostly lower order animals, this natural response has been developed into a powerful tool for the investigation of gene function (Elbashir S M, et al., Genes Dev, 2001, 15:188-200; Hammond S M, et al., Nat Rev Genet., 2001, 2:110-119). More recently it was discovered that introducing synthetic 21-nucleotide dsRNA duplexes into mammalian cells could efficiently silence gene expression. Although the precise mechanism is still unclear, RNAi offers a new way to inactivate genes of interest. In particular, for the treatment of neoplastic disorders (cancer), RNAi provides a potential new approach to modulate (e.g., reduce) the expression of certain genes, e.g., an anti-apoptotic molecule, a growth factor, a growth factor receptor, a mitotic spindle protein, a cell cycle protein, an angiogenic factor, an oncogene, an intracellular signal transducer, a molecular chaperone, and combinations thereof.
One such target is the Eg5 gene which produces the microtubule-associated protein, Eg5 (Kapoor, T. M., et al., J. Cell Biol., 2001, 154, 1125-1133; Garrett, S. et al., Curr Biol., 2003, 13, R810-812; Cassimeris, L., et al., Curr Issues Mol. Biol., 2003, 5, 99-112; Houliston, E., et al., Dev. Biol. 1994, 164, 147-159). Generally, due to the clinical success seen with antimitotics in cancer therapy (e.g. taxanes), other proteins that that are also involved in the mitotic machinery, such as Eg5, have become desirable targets as next generation anti-cancer therapeutics. The mitotic kinesin, Eg5, contains an N-terminal motor domain which generates force along the microtubule, moving Eg5 to the microtubule plus end. During interphase in most epithelial cells, the plus ends of microtubules are oriented toward the plasma membrane while the minus ends are facing toward the nucleus. Upon entry into mitosis, microtubule plus ends reorient toward the chromosomes, while the minus ends are anchored at the spindle poles, forming a bipolar spindle. The homotetrameric structure of Eg5 has its motor domains arranged at two ends of a dumbbell such that it can bind and push apart spindle microtubules and generate and outward-directed force pushing spindle poles apart (Sawin, K. E., et al., Prod Natl. Acad. Sci. USA, 1995, 92, 4289-4293; Kapoor, T. M., et al., J Cell Biol., 2000, 150, 975-988; Gaglio, T., et al., J Cell Biol., 1996, 135, 399-414). Thus, Eg5 is critical for proper spindle formation during the mitotic process. Disruption of the process leads to activation of spindle assembly checkpoint, the major cell cycle control mechanism which prevents the cell undergoing mitosis to progress to anaphase and leads to cell cycle arrest. Inhibition of the production of the Eg5 protein, which is only expressed during mitosis, results in the induction of cancer cell apoptosis through a unique mechanism. In view of the important role of Eg5 has in the mitotic process, Eg5 has become an attractive therapeutic target for rapidly dividing cells, in particular, in cancer therapy.
The first small molecule inhibitors of Eg5 was identified in a phenotype-based screen and has been termed Monastrol, because of the formation of monoastral spindles due to Eg5 inhibition seen in cells treated with Monastrol (Mayer, T. U., et al., Science, 1999, 286, 971-974). Other small molecule inhibitors of Eg5 have been discovered since and are currently under development (Sakowicz, R., et al., Cancer Res., 2004, 64, 3276-3280; Hotha, S. et al., Angew Chem. Intl. Ed. Engl., 2003, 42, 2379-2382). However, recent literature publications suggest that certain cancer cell lines, e.g., HT-29 colorectal cancer cells, are resistant toward small molecule Eg5 inhibitors, and thus suggest that small molecule Eg5 inhibitors may have be of more limited clinical use than previously thought.
A second target is the EGFR gene that encodes for the epidermal growth factor receptor (EGFR), a glycoprotein with a molecular weight of 170,000 to 180,000. EGFR is an intrinsic tyrosine-specific protein kinase, which is stimulated upon epidermal growth factor (EGF) binding. The known downstream effectors of EGFR include PI3-K, RAS-RAF-MAPK P44/P42, and protein kinase C signaling pathways. EGFR signaling involved in cell growth, angiogenesis, DNA repair, and autocrine growth regulation in a wide spectrum of human cancer cells (Wakeling A E., Curr Opin Pharmacol 2002, 2:382-387). Therefore, it has recently emerged as an innovative target for the development of new cancer therapy. Recently, a monoclonal antibody against EGFR called cetuximab has been developed. It has shown excellent clinical effects for the treatment of lung and head and neck cancer in a clinical trial in humans (Shin et al., Clin Cancer Res, 2001, 7:1204-1213). Other small chemical inhibitors, such as ZD-1839 have also been developed and demonstrated anti-tumor effects in in vitro and in vivo (Shawver L K, et al., Cancer Cell 2002, 1:117-123). However, clinical use of ZD-1839 in humans has not been very successful. (Baselga Eur J Cancer 2001, 37:S16-22).
A third target is the XIAP (X-linked inhibitor of apoptosis protein) which is a member of the mammalian IAP gene family and encodes for the X-linked IAP (XIAP) protein. The anti-apoptotic function of XIAP is executed, at least in part, by inhibition of caspase-3 and caspase-7, two principal effectors of apoptosis. XIAP protein plays a critical role in regulating programmed cell death by suppressing activation of downstream caspase-3 and caspase-7. Interestingly, in pre-cancerous and cancerous cells, it is believed that expression or overexpression of XIAPs makes it difficult for cancer cells to eliminate themselves, instead allowing them to proliferate, metastasize and accumulate additional oncogenic mutations. Inhibition of XIAP activity using anti-sense oligonucleotides has demonstrates anti-tumor activity in human tumor xenograft animal model.
In view of the above, there is a need for compositions and methods for modulating the expression of genes involved in cancer. The present invention addresses these and other needs.